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Hereng T.H.,Spermatech AS | Backe P.H.,University of Oslo | Kahmann J.,Evotec | Scheich C.,Evotec | And 4 more authors.
Journal of Structural Biology | Year: 2012

Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named Cα2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo Cα2 to 2.1 å. Apo Cα2 displays an open conformation similar to the well-characterized apo structure of murine Cα1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13° in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for Cα2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of Cα1. Human Cα2 and Cα1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of Cα1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. Cα2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that Cα2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization. © 2012 Elsevier Inc.


Aksaas A.K.,University of Oslo | Larsen A.C.V.,University of Oslo | Rogne M.,University of Oslo | Rosendal K.,Spermatech AS | And 2 more authors.
Journal of Molecular Signaling | Year: 2011

Background: Post-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications of a direct interaction between RNA and PKA. Based on this we speculate if the specificity of the C subunit in regulating pre-mRNA splicing may be mediated indirectly through other nuclear proteins.Results: Using yeast two-hybrid screening with the PKA C subunit Cbeta2 as bait, we identified the G-patch domain and KOW motifs-containing protein (GPKOW), also known as the T54 protein or MOS2 homolog, as an interaction partner for Cbeta2. We demonstrate that GPKOW, which contains one G-patch domain and two KOW motifs, is a nuclear RNA-binding protein conserved between species. GPKOW contains two sites that are phosphorylated by PKA in vitro. By RNA immunoprecipitation and site directed mutagenesis of the PKA phosphorylation sites we revealed that GPKOW binds RNA in vivo in a PKA sensitive fashion.Conclusion: GPKOW is a RNA-binding protein that binds RNA in a PKA regulated fashion. Together with our previous results demonstrating that PKA regulates pre-mRNA splicing, our results suggest that PKA phosphorylation is involved in regulating RNA processing at several steps. © 2011 Aksaas et al; licensee BioMed Central Ltd.


Hereng T.H.,Spermatech AS | Cederkvist F.H.,University of Oslo | Eide L.,University of Oslo | Jahnsen T.,Spermatech AS | And 4 more authors.
Human Reproduction | Year: 2011

Background: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility.Methods: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry.Results: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the Results: , indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50 in the presence of exogenous pyruvate. Moreover, all consumed 13C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. Conclusions: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our Results: . As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female. © 2011 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.


Hereng T.H.,Spermatech AS | Elgstoen K.B.P.,University of Oslo | Eide L.,University of Oslo | Rosendal K.R.,Spermatech AS | And 2 more authors.
Human Reproduction | Year: 2014

STUDY QUESTION: Do the known capacitating agents HCO3 - and serum albumin regulate the generation of ATP required for sperm motility and capacitation? SUMMARY ANSWER: Serum albumin and HCO 3 - seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa. WHAT IS KNOWN ALREADY: Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation. STUDY DESIGN, SIZE, DURATION: Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO3 - and serum albumin for up to 4 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO3 - were analysed by metabolic tracing of 13C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions. MAIN RESULTS AND THE ROLE OF CHANCE: We found that HCO3 - significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO3 --induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl-and methyl-β-cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca2+ channel CatSper. Finally, we demonstrated that neither HCO3 - nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO3 -.LIMITATIONS, REASONS FOR CAUTIONGreat variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species. WIDER IMPLICATIONS OF THE FINDINGS: It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO 3 - stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques. © 2014 The Author.


PubMed | Spermatech AS
Type: Journal Article | Journal: Journal of structural biology | Year: 2012

Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named C2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo C2 to 2.1 . Apo C2 displays an open conformation similar to the well-characterized apo structure of murine C1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13 in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for C2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of C1. Human C2 and C1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of C1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. C2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that C2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization.


PubMed | Spermatech AS
Type: Journal Article | Journal: Human reproduction (Oxford, England) | Year: 2014

Do the known capacitating agents HCO(3)(-) and serum albumin regulate the generation of ATP required for sperm motility and capacitation?Serum albumin and HCO(3)(-) seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa.Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation.Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO(3)(-) and serum albumin for up to 4 h.Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO(3)(-) were analysed by metabolic tracing of (13)C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions.We found that HCO(3)(-) significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO(3)(-)-induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl- and methyl--cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca(2+) channel CatSper. Finally, we demonstrated that neither HCO(3)(-) nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO(3)(-).Great variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species.It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO(3)(-) stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques.The work was funded by Spermatech AS, The University of Oslo and the Research Council of Norway.T.H.H. and K.R.R. are employees at Spermatech. B.S.S is a shareholder in Spermatech.


PubMed | Spermatech AS
Type: Journal Article | Journal: Human reproduction (Oxford, England) | Year: 2011

There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility.Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry.The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle.Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

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